1. Field of the Invention
The present invention relates to a method of determining the total viable bacteria count of Enterobacteriaceae which is capable to or forming spreading colonies or spreaders and of causing food poisoning, sporeforming bacteria which are difficult to destroy using ordinary heat treatment during food processing, and other bacteria in food at the same time, and to a powdery medium for determining the viable bacterial counts of food, whereby the bacteria count can be determined without the formation of a spreading colony.
2. Description of the Prior Art
From the present technical level of microbiology, it would appear to be a simple matter to determine the viable bacteria count of food.
However, when an agar medium, which is conventionally used in practice, is used for determining the viable bacteria count by forming colonies on a plate of said agar medium and counting the numbers of viable bacterium, it is often experienced that certain kinds of bacterium present undergo an extraordinary spreading of their colonies. Typically, the neighbouring colonies contact each other and, in the extreme case, the colonies spread over all the surface of the medium plate which makes the exact count of number of colonies present impossible.
The "Spreading of Colonies" or the formation of "Spreaders" can be avoided to some degree by completely drying the surface of medium plate so that no water drops are remain at the time of incubation.
But in some bacteria it is difficult to prevent "Spreading of Colonies" of these bacteria even if the drying is complete, and only a few colonies spread all over the surface of medium plate. When the number of colonies becomes greater than 100 per each plate, spreading can hardly be prevented.
Bacteria liable to form spreading colonies include Proteus, Escherichia, Aerobacter, Serratia and the like which belong to Enterobacteriaceae (Bacteriological Reviews, 36, 478, 1972 and Bergey's Manual of Determinative Bacteriology, page 322, 7th Edition, The Wiliam & Wilkins Company, Baltimore, U.S.A., 1957). All of these bacteria participate in spoilage of food and in the outbreak of food poisoning.
However, bacteria which form spreading colonies most remarkably are sporeformers. The sporeformers have two kinds of genus, that is, aerobic Bacillus and anaerobic Clostridium. These bacteria have the characteristic of easily forming spreading colonies and include a pathogenic species such as Bacillus anthracis and Clostridium botulinum and many other bacteria which survive through the heating and pasteurizing step of food processing and cause the deterioration food in distribution.
The bacteria capable of forming spreading colonies, as described above, are widely distributed in nature and have a wide temperature range of growth, and, therefore, from a viewpoint of food hygiene, it is an important problem to determine the viable count of these bacteria in food exactly. However, the conventional method for the determination or the viable bacteria count does not take any steps to prevent the formation of spreading colonies at the time of incubation of bacteria. Therefore, on page 48 of "Standard Methods for the Examination of Dairy Products" 12th edition published by the American Public Health Association, Inc. New York, (1967), it is stipulated that if spreaders occur on the plate(s) selected, one counts the colonies on representative portions thereof only when (1) colonies are well distributed in spreader-free areas and (2) the area covered by spreader(s), including total repressed-growth area, if any, does not exceed one-half of plate. However, in case of using the conventional medium, errors are in any case unavoidable and it is too much to say that the counts in a case where a spreading colony is actually formed is only an estimated value.
As a result of studying the difficulty of determining viable counts of food-contaminating bacteria which form spreading colonies, the present inventors have found that 2, 3, 5-triphenyl-tetrazolium chloride (hereinafter abbreviated to TTC) can prevent the spreading of the bacteria and the formation of spreading colonies without inhibiting the growth of bacterium not forming a spreading colony. The inventors have further found that an incubation plate which contains no spreading colonies is obtained by using said compound, TTC, as the component of a medium conventionally used for the determination of viable bacterial counts of food so that a more exact determination of viable bacterial counts becomes possible.
An aqueous solution of TTC is colorless but it turns red by the reduction reaction due to the convertion of TTC to insoluble formozan. TTC has hitherto been used as an indicator for testing the growth of bacteria by utilizing this characteristic. For example, TTC has been used for testing antibiotics in cow's milk. (Neal et al, Journal of Dairy Science, 38, 629, 1955) and judging for the quality of pasteurized milk (Broitman et al, Journal of Milk and Food Technology, 19, 63, 1956). In these reports TTC is employed as an indicator utilizing the phenomenon that TTC is reduced by the growth of bacteria in cow's milk medium and colored.
On the other hand to CVT medium reported by Olsen, which is a selective medium for psychrophilic bacterium, (Journal of Dairy Science, 46, 362, 1963) and EF medium placed on the market by Nissui Pharmaceutical Co., Ltd., which is a medium for the identification of Enterococcus, is added 0.005% and 0.015%, respectively, of TTC. Either of these media are used for classification of bacteria according to the depth of color tone of growth colony caused by the difference of the reducing activity of various bacteria for TTC.
Also, the Japanese Patent Public Disclosure Gazette No. 125081/75 describes a method of easily detecting coliform bacilli group in a sample by directly contacting a filter paper strip impregnated with an aqueous solution of TTC and liquefied agar medium to the sample. However, in this method TTC is used as a selective agent for inhibiting the growth of fungus and gram-positive bacteria, and for the purpose of determining total viable bacterial counts of the sample, the filter paper strip impregnated with TTC and medium cannot be utilized.
As described above, TTC has hitherto been employed for identification of specific substance or isolation and classification of specific bacterium in a specific medium, but has not been used for preventing the formation of spreading colonies, as a component of medium for determining the viable bacterial count of food.